The Ras family of small guanine 5'-triphosphatases (GTPases) comprise a group of molecular switches that link receptors on the cell surface to signaling pathways that regulate cell proliferation and differentiation. Ras proteins are highly conserved among eukaryotic species. Mutant forms of Ras that are locked in the guanine 5'-triphosphate (GTP)-bound state, and thus continuously drive cell proliferation by interacting with their target proteins, are found in 30 percent of human tumors. This application describes a novel protein in budding yeast that appears to serve two separate functions, despite the fact that it is only 68 amino acids in length. The gene encoding this protein, designated RIN1 (for Ras inhibitor 1) behaves genetically as an inhibitor of Ras, and the Rin1 protein associates in vivo with GTP-bound Ras in a manner that suggests it competes for the same binding site as Ras target proteins. Because Rin1 resides in the membrane of the ER, rather than on the cell surface with the majority of Ras, it is hypothesized that one function of Rin1 is to maintain Ras in an inactive state during its posttranslational modification in the ER. The long-term objective of this project is to develop a form of Rin1 that effectively inhibits GTP-bound Ras on the cell surface for the purpose of treating Ras-involved malignancies. The second function of Rin1 is in the processing or transport of glycosyl-phosphatidylinositol (GPI)-anchored proteins from the ER. It is hypothesized that this function is carried out through association with a second member of the Ras family that is involved in protein secretion. The specific aims of this project are: 1) To understand the membrane topology of Rin1, and to identify the Ras-interaction region. This is the first step toward developing a form of Rin1 that is optimized for Ras inhibition at the cell surface. 2) To understand the function of Rin1 in GPI-protein anchoring or secretion. This will involve identification of the second target of Rin1. It is also likely that this aim will contribute to the new area of protein sorting in the ER. 3) To determine if yeast Ras can signal from the ER, and to create a more effective Rin1-based inhibitor of Ras. This aim is focused on assessing the function of Ras expressed in the ER, and the function of the Ras-interacting domain of Rin1 expressed at the cell surface. These experiments will help to test the applicant's model of Rin1 function as an inhibitor of Ras signaling in the ER. [unreadable] [unreadable]